Saturday, March 28, 2015

Quasi Species: Defending my follow up

Greetings

I had recently posted a post on how do you explain a lot of mutations in sequences from viral outbreaks such as that of Ebola and Influenza (Link). Evidently, some people have misunderstood what I mean to say and there was an aggressive debate on this issue. I was referred to an article in nature news (Link), and my previous blogs on Ebola. I did mention that a lot of mutations are seen in this outbreak, about 300 plus mutations (Link), but I never argued that we are at an accelerated mutation rate and we are evolving more pathogenic strains. It took me sometime to convince the person on other side. Here's my defense argument in a summary. Coincidentally there is a paper right on time, which I can cite.

A new paper in science by Sow etal, has analyzed sequences from the context of Ebola outbreak. The study has analyzed a large set of sequences and determined that nucleotide substitution rate ( 9.6 × 10–4 substitutions per site per year), which is consistent with previous outbreaks. The paper concludes that the virus is not mutating any faster than what it is supposed to be nor is more virulent. As summed up by Sabeti, "We have always been clear that our study only provided data for what everyone already knows that viruses mutate,The data never said that, but it was the way people interpreted the data". 

But then the question remains, why are we seeing so many mutations. As I have already argued, virus are a quasi species. More time spent, more are the mutations. Ebola has spread over a very wide geographical area than any other previous outbreak. So in comparison there was a lot of time to have sequence changes. The dendrogram and mutation data depends on the time frame of samples being sequenced, and the number of sequence. This exactly is my point. Virus mutation accumulates overtime. Moreover, the rate of mutation is not the paramount question. It is acquiring a mutation that really translates. And as the paper finds, there are several mutations but nothing that has affected the pathogenesis or significantly of immune interest. Quoting from science, "The new data adds yet more confidence that a vaccine strategy should work he says, because the virus appears fairly stable. So, good news."

ResearchBlogging.org
Hoenen, T., Safronetz, D., Groseth, A., Wollenberg, K., Koita, O., Diarra, B., Fall, I., Haidara, F., Diallo, F., Sanogo, M., Sarro, Y., Kone, A., Togo, A., Traore, A., Kodio, M., Dosseh, A., Rosenke, K., de Wit, E., Feldmann, F., Ebihara, H., Munster, V., Zoon, K., Feldmann, H., & Sow, S. (2015). Mutation rate and genotype variation of Ebola virus from Mali case sequences Science DOI: 10.1126/science.aaa5646

Vogel, G. (2015). A reassuring snapshot of Ebola Science, 347 (6229), 1407-1407 DOI: 10.1126/science.347.6229.1407

Thursday, March 26, 2015

Cerebral malaria- In short

Greetings

I have previously written some posts on malaria, the challenges in treating and vaccination strategies. Research continues on these fields with significant progress being made. However, isn't it interesting that I never talked about cerebral malaria. I couldn't find a good recent statistics of malaria burden, but the reports indicate that there is a significant decrease in number of cases on a global scale. However, if you consider the parasitic infections, malaria stands taller than anybody else. Of the people infected with P falciparum, roughly 10% have cerebral malaria (CM) depending on the geography. Nearly 575,000 children in Africa develop cerebral malaria annually. On a global scale CM is a significant problem.

The mechanism of CM is not a well understood phenomenon. Studies to understand the pathogenesis have been carried in murine models involving C57BL/6 or CBA mice infected with P berghei ANKA. Currently, It is the best models to study CM pathogenesis. In models, it has been shown that cytoadherence of parasite and disruption of blood brain barrier, leads to hemorrhages and necrosis of the surrounding tissues and cerebral edema. The increased brain volume which leads to compression and raised intra cranial pressure is probably the reason for fatality. In a recent paper in NEJM, this has been studied using MRI scans and confirmed validating the previous understanding. As Dr Taylor puts it, "Because we know now that the brain swelling is what causes death, we can work to find new treatments. The next step is to identify what's causing the swelling and then develop treatments targeting those causes. It's also possible that using ventilators to keep the children breathing until the swelling subsides might save lives, but ventilators are few and far between in Africa at the moment".

Fig 1: PfEMP1 Structure. Source
Perhaps the most focused part of studying the CM is cytoadherence. Cytoadherence is the phenomenon of sticking together of RBC to the vascular membrane. The best described adhesin studied in P falciparum is PfEMP1 (P. falciparum erythrocyte membrane protein 1). The protein is encoded by "var" gene and large transmembrane protein (200–350 kDa) comprising several external Duffy-binding-like domains. The structure of PfEMP1 includes a large extracellular N-terminal domain, a transmembrane region and a C-terminal intracellular domain. The extracellular domain is highly variable and at least a 50 variants are known. The C terminal anchors protein to the RBC membrane and is conserved structure. The protein is encoded by "var" gene and large transmembrane protein (200–350 kDa) comprising several external Duffy-binding-like. The PfEMP1, is produced by the parasite and inserted to the RBC membrane by protein trafficking. The inserted protein is called as a knob.

Fig 2: Receptor interactions. Source
The PfEMP1 has an important role of immune evasion. Studies have shown that the knob has a positive charge and hence better electrostatic interaction with negatively charged endothelial plasma membranes and receptors. Cytoadherence prevents malarial parasites from being recognized and cleared off the circulation by the spleen. In addition the variance adds to immune evasion. There are several receptor interactions in the picture, the most important include- CD36, TSP, CD54, ICAM-1, VCAM-1 and P- selectin. It has also been speculated that the rosetting (clumping), slows the movement and oxygenation of RBC which provides a anaerobic rich growth environment for parasite.

Fig 3: Model for CM. Source
The second important step in the process is sequestration of parasitised RBC (PRBC). The PRBC accumulate in microvessels of various organs. In the brain, the most problematic is in brain especially cerebrum, cerebellum and medulla oblongata. The plugging of microvessels leads to hypoxia which stimulates further parasitic growth. Subsequently, this event leads to inflammation, with major role played by Pro-inflammatory cytokines and reactive oxygen species. The events lead to breakdown of BBB (Blood brain barrier), allowing uncontrolled movement of fluid. This leads to the hypertension and raised Intra cranial pressure. The increased pressure leads to neuronal compression and damage. In events of medulla oblongata being effected, vital functions may decline leading to death.

The model is well explained in a figure by Frevert and Nacer. See Fig 3

As you see, the pathogenesis of CM is fairly understood. So the new study in NEJM doesn't surprise me at all. The only new thing is study they did an MRI. As Dr. Taylor comments. "I wanted to say to the parasite 'Ha! You never thought we'd get an MRI, did you?'"

ResearchBlogging.org
Bagot S, Idrissa Boubou M, Campino S, Behrschmidt C, Gorgette O, Guénet JL, Penha-Gonçalves C, Mazier D, Pied S, & Cazenave PA (2002). Susceptibility to experimental cerebral malaria induced by Plasmodium berghei ANKA in inbred mouse strains recently derived from wild stock. Infection and immunity, 70 (4), 2049-56 PMID: 11895970

Seydel KB, Kampondeni SD, Valim C, Potchen MJ, Milner DA, Muwalo FW, Birbeck GL, Bradley WG, Fox LL, Glover SJ, Hammond CA, Heyderman RS, Chilingulo CA, Molyneux ME, & Taylor TE (2015). Brain swelling and death in children with cerebral malaria. The New England journal of medicine, 372 (12), 1126-37 PMID: 25785970

McMillan PJ, Millet C, Batinovic S, Maiorca M, Hanssen E, Kenny S, Muhle RA, Melcher M, Fidock DA, Smith JD, Dixon MW, & Tilley L (2013). Spatial and temporal mapping of the PfEMP1 export pathway in Plasmodium falciparum. Cellular microbiology, 15 (8), 1401-18 PMID: 23421990

Frevert U, & Nacer A (2014). Fatal cerebral malaria: a venous efflux problem. Frontiers in cellular and infection microbiology, 4 PMID: 25414834

Monday, March 23, 2015

Quasi Species- Discussion

Greetings

I have been posting regularly on "Infection Outbreaks". In my previous post, I talked about Indian Influenza outbreak that has been concerning and received some attention. With a publication claiming there is a mutation of interest which may make the H1N1 more pathogenic but Indian studies showing that there isn't such a scenario has created a duality (Link). But what causes emergence of Rapid mutation in viruses like that of Influenza? That's an interesting question to ask. May be the question deserves a blog space here. I have previously talked about mutation (Link here and here).

Let us take the example of Q-beta phage which first demonstrated the depth of genetic variance in virus. Let me quote from a post in virology blog (Link). "A Q-beta phage population is in a dynamic equilibrium with viral mutants arising at a high rate on the one hand, and being strongly selected against on the other. The genome of Q-beta cannot be described as a defined unique structure, but rather as a weighted average of a large number of different individual sequences". This dynamism in the virus population within the species. There is misinterpretation that virus have a excessive mutation rate. It is true that there is about one magnitude difference in mutation rate for viral genomes, thanks to the lower fidelity of viral replication enzyme. But its the massive replication that leads the way.

Let me cite Influenza as an example, since it is of great interest. In experimental conditions, Influenza A virus begins producing a progeny aprox 6 h after infection of cell, and continue to do so for another 5 h. A single infected cell produces 22 new productive infections on average. Mathematically, even if you had consider that there is a mutation once in a 1000 replication, at the end of a week, you would have enough genetically diverse types. You also have to consider fitness cost. Not all mutations are good for the virus to have. So many of them would simply vanish of. So, if you had take a sample of viral population in circulation, it is almost imperative that you had find some mutations. To have a clear idea of how much the mutation has penetrated, you need to have lot of sequences.

This concept presents with an often overlooked point. For many viral infections seroconversions and rapid accumulation of mutation is a common phenomenon. This doesn't mean things suddenly happened that way overnight. For example, Ebola is a case of recent interest. In ever case there would be a large set of viruses that have slightly different mutation. However, only the one's which resist the immune attack and are successful would be predominantly present in the sample (Fitness cost), and studies will yield the same. That's the whole point of constructing dendrograms. It lets you elute the dynamism. I recently had a discussion about this, and I thought its worth posting the same here. See Fig 1

ResearchBlogging.org
Baccam, P., Beauchemin, C., Macken, C., Hayden, F., & Perelson, A. (2006). Kinetics of Influenza A Virus Infection in Humans Journal of Virology, 80 (15), 7590-7599 DOI: 10.1128/JVI.01623-05

Nobusawa, E., & Sato, K. (2006). Comparison of the Mutation Rates of Human Influenza A and B Viruses Journal of Virology, 80 (7), 3675-3678 DOI: 10.1128/JVI.80.7.3675-3678.2006

Sanjuan, R., Nebot, M., Chirico, N., Mansky, L., & Belshaw, R. (2010). Viral Mutation Rates Journal of Virology, 84 (19), 9733-9748 DOI: 10.1128/JVI.00694-10

Friday, March 13, 2015

Indian Influenza Outbreak: 2015- Follow up

Greetings

Fig 1: Current estimates of swine flu outbreak.
   I have previously blogged about increased number of Influenza cases being reported in India (Link). I don't have a clear official data on the numbers, and so in this case I will rely on media reports which mayn't be absolute. As of on March 11, 2015 27,234 cases and 1,537 deaths have been reported from India. The worst effected states include Telangana, Rajasthan and Gujarat. From the press release, as on March 12, 2015 a total of 1925 samples have tested positive so far with 69 deaths in Telangana. Fig 1 gives an estimate of current cases in heavily affected regions.

At the time of writing my previous post there was virtually no data available on the molecular epidemiology of current outbreak. There is a great demand to know the details of the virus especially its sequence. NIV has sequenced 4 genomes and reported that it is no different from known H1N1 stable strain. However, a stir of debate has come out with a publication claiming Indian strains carry new mutations in the hemagglutinin protein that are known to make the virus more virulent. The reported mutations of concern are D225, which has been linked with increased disease severity and T200A which allows hemagglutinin to bind more strongly to glycan receptors.On the other hand the health ministry states, ""The virus has not undergone any major mutation so as to make it more virulent and nor has the HINI virus become resistant to the mainline drug Tamiflu".

Now here is the catch that has not been prominent in the headlines everywhere. The published research study is based on isolates from 2014 strains. NIV has sequenced less than a handful and there is no real time monitoring data. It is very hard to draw a conclusion form both sides of the argument. There were a few reported case of oseltamivir-resistant H1N1 reported from Hokkaido, Northern China, U.S and Australia. The past outbreaks of resistant flu havent appeared in literature and probably safe to say its not a potential problem yet. To make any conclusive statements we need more sequences. In fact a lot more sequence. In addition, India should consider active influenza vaccination like that of other countries.

As Alfred H. Caspary puts it, "We're really caught between a rock and a hard place, with little information and a lot of misinformation. When you do real-time surveillance, get organized, and deposit these sequences, then you can come up with a better strategy to respond to the virus." Source

And for those people who are worried of this outbreak, simple sanitary measures and hygiene will go a long way. Influenza is not as dangerous as it is posed to be.

ResearchBlogging.org
Kannan Tharakaraman, Ram Sasisekharan (2015). Influenza Surveillance: 2014–2015 H1N1 “Swine”-Derived Influenza Viruses from India Cell Host and Microbe, 17 (3) : doi:10.1016/j.chom.2015.02.019

Monday, March 09, 2015

Toll Like Receptors

Greetings

Cells are an amazing bag of self sustaining chemicals, if I could put it that way. This statement not only means it is inherently meant to power itself, it should also be able to defend itself from other's who means to steal it. In a post long ago (Link), I had talked a little bit about sensing of viral DNA in context with STING (Simulator of Interferon genes) pathway. But before the cell decides, they need to trigger a reaction, a signal has to arrive that it is warranted. In other words sensor messenger's that alert to the need of immunity.

The problem in having a innate immune sensor that detects any invader is that there is a huge number of possible signatures. It is not possible for cell to develop every kind of sensor. Instead evolution has selected for sensors based on conserved biological signatures. Such signatures are referred are called as pathogen-associated molecular patterns (PAMPs). There are several types of sensors (somebody needs to write a book on it). Someone asked me if I could put up some basics on TLR's. So here it is.

Toll was originally studied in Drosophila. The first human Toll-like receptor was described by Nomura etal in 1994 and chromosomal location identified by Taguchi etal in 1996 When a similar homologue was found in humans, it was called as Toll like receptor, and the name stands true till date. In the Drosophila genome, 9 Toll genes are noted. In Drosophila, Toll receptor is activated when proteolytically cleaved by a ligand Spatzle binds to the receptor, eventually leading to the activation of the NF-κB factors and production of antimicrobial factors. 

Table 1: TIR protein sub-types.
Human TLRs are 700–1100 amino acids long transmemebrane proteins with Leucine-rich repeat extracellular domain, a transmembrane region, and an internal cytoplasmic domain. Cytoplasmic domain is similar to IL-1 receptor family, and thus termed as Toll/IL-1 receptor (TIR) domain. The TIR domain is divisible into 3 sets- Type I to III. See Table 1.

It would be of no use to the reader to write a long essay about all the TLR's. So I simply summarized everything into a table. See Table 2

Table 2: Summary of Toll Like receptor- basics

ResearchBlogging.org
Valanne, S., Wang, J., & Ramet, M. (2011). The Drosophila Toll Signaling Pathway The Journal of Immunology, 186 (2), 649-656 DOI: 10.4049/jimmunol.1002302

Takeda, K. (2005). Toll-like Receptors and their Adaptors in Innate Immunity Current Medicinal Chemistry - Anti-Inflammatory & Anti-Allergy Agents, 4 (1), 3-11 DOI: 10.2174/1568014053005336

Kawasaki, T., & Kawai, T. (2014). Toll-Like Receptor Signaling Pathways Frontiers in Immunology, 5 DOI: 10.3389/fimmu.2014.00461

Tuesday, March 03, 2015

A thought on Microbiome

Greetings

Beyond everything else, I first want to express a great deal of happiness for posing me as one of the top Indian Young Scientific Contributors, in field of microbiology, in the magazine Micrographia Today (Link). Second, The past week I had the pleasure of interacting with Eric Lander, who visited the campus to interact with students. Also his later talk in TNQ series (Link) was fascinating. The lack of time had not allowed for a deep discussion, but his perspectives in thinking about problems in genetics led me to rethink certain aspects of microbiome.

Fig 1:Outcome depends on
Multiple interactions.
I have already talked about the microbiome in my previous posts (Link here and here). I need to digress a little bit here to see where the argument is heading. A lot of understanding in science is contextual basis and the difference between good and bad is often very blurred. For example, Obesity is one of the greatest challenge in current medicine. The situation is probably multi-factorial and genes have a lot of role to be played here. So would you designate the gene as bad. The tendency is to say so. The point is its not. There is nothing called bad and good genes. It depends on context. I have got into lot of arguments on this point earlier. Since Eric affirmed in this case my thinking is right, I will go ahead with argument. In a time when humans where evolving, food was scarce having a gene that makes you obese was actually a bonus. The situation has changed only in past 200-400 years, a time too small to evolve out of it.

Now put the above story in context with microbiome, and the picture becomes a little clear. My thinking has been influenced by a post in small things considered (Link), as I have quoted in my previous post. There is no good or bad microbiome. In Eric's talk, he posed the difficulty of understanding the microbiome. Does the microbiome influence the condition or is it the other way around. In other words what is the direction of effect. This requires a lot of complicated experiments to understand, however there are some interesting findings. For example, obesity can be transferred in mouse models (Link). A more recent story of a woman gaining weight after FTM procedure has opened a panel of discussion. Evidence is emerging as to association with other factors such as wound healing. The studies hint that microbiome can have a serious effect. But to extrapolate this finding to every condition is exploitation of the concept.

In the course of thinking it occurred to me that diversity in microbiome may explain some findings. I'm not sure if I conveyed the idea right during discussion. So let me elaborate my thinking, though I must warn that these are purely my thoughts of projection. As an example, I recently blogged about C scindens and its effect on C difficile (Link). There are a lot more examples of one organism countering the strength of other which is an important part of residential flora- competence. So it is very attractive to speculate having a more diverse population control each other. On the other hand, in some conditions the diversity is suppressed allowing some strains to predominate which may in-turn influence the normal process to a higher degree tipping of the balance. Thinking along these lines seems to explain at least some of the concepts. This also allows variability of the microbiome to be accounted for that is normally seen.

Would like to know what thoughts of the readers are.

ResearchBlogging.org
Alang, N., & Kelly, C. (2015). Weight Gain After Fecal Microbiota Transplantation Open Forum Infectious Diseases, 2 (1) DOI: 10.1093/ofid/ofv004

Weil, A., & Hohmann, E. (2015). Fecal Microbiota Transplant: Benefits and Risks Open Forum Infectious Diseases, 2 (1) DOI: 10.1093/ofid/ofv005

Grice EA, & Segre JA (2012). Interaction of the microbiome with the innate immune response in chronic wounds. Advances in experimental medicine and biology, 946, 55-68 PMID: 21948362

Blaser MJ (2014). The microbiome revolution. The Journal of clinical investigation, 124 (10), 4162-5 PMID: 25271724